IKKβ is required for Bcl-2-mediated NF-κB activation in ventricular myocytes

The transcription factor nuclear factor kappaB (NF-kappaB) is regulated by cytoplasmic inhibitor IkappaBalpha. An integral step in the activation of NF-kappaB involves the phosphorylation and degradation of IkappaBalpha. We have previously reported that IkappaBalpha activity is diminished in ventricular myocytes expressing Bcl-2 (de Moissac, D., Zheng, H., and Kirshenbaum, L. A. (1999) J. Biol. Chem. 274, 29505-29509). The underlying mechanism by which Bcl-2 activates NF-kappaB is undefined. In view of growing evidence that the IkappaB kinases (IKKs), notably IKKbeta, are involved in signal induced phosphorylation of IkappaBalpha, we ascertained whether IKKbeta is necessary and sufficient for Bcl-2 mediated NF-kappaB activation. Here we demonstrate that expression of Bcl-2 in ventricular myocytes resulted in an increase in NF-kappaB-dependent DNA binding, NF-kappaB gene transcription and reduced IkappaBalpha levels. An increase in the IKKbeta kinase activity was observed in cells expressing full-length Bcl-2 but not in cells expressing the BH4 deletion mutant of Bcl-2 (DeltaBH4; residues 10-30). Catalytically inactive mutants of IKKbeta, but not IKKalpha, suppressed Bcl-2-mediated IkappaBalpha phosphorylation and NF-kappaB activation. Transfection of human embryonic 293 cells with a kinase-defective Raf-1 or a kinase-defective mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-1 (MEKK-1) suppressed Bcl2-mediated IKKbeta activity and NF-kappaB activation. Further, Bcl-2-mediated NF-kappaB activity was impaired in nullizygous mouse embryonic fibroblasts deficient for IKKbeta. In this report, we provide the first direct evidence that Bcl-2 activates NF-kappaB by a signaling mechanism that involves Raf-1/MEKK-1 mediated activation of IKKbeta.